Many immune-mediated diseases-infectious diseases like HIV and autoimmune diseases like multiple sclerosis or diabetes-mediate pathology in specific tissues, yet most of our knowledge about them has resulted from studying cells circulating in blood. These cells have been a convenient proxy because blood is the most accessible compartment and the number of cells recovered can be large. Increasing evidence suggests, however, that the biology of diseases in affected tissues can vary substantially from that in the blood, and understanding these differences may be critical to develop new drugs, vaccines, and diagnostics to improve patient care. The significant heterogeneities among cells resident in tissues necessitates characterizing such samples with single-cell resolution, but existing technologies routinely employed by clinical immunologists (flow cytometry, ELISpot) typically require an excess of cells to use for analysis. Their inefficiencies have hindered the ability to pursue science understanding the human biology of diseases and treatments in tissues because biopsies yield very few cells. This research will optimize, validate, and deploy a unique nanowell-based platform to address this unmet need for characterizing single cells from clinical biopsies with minimal manipulations. The project is a collaboration amongst: the Love and Lauffenburger Labs (MIT) with expertise in applying microfabricated technologies to resolve single-cell heterogeneities and in developing computational tools for analyzing such data; the Kwon and Walker Labs (Ragon Institute) with expertise on the clinical immunology of HIV and vaccines; the Mesirov and Wong Labs (Broad Institute) with expertise in developing software tools for data analysis and means of visualizing complex data; and the Roederer Lab (NIH VRC) with expertise in single-cell technologies for characterizing immunophenotypes and gene expression. Together, this interdisciplinary team spanning engineering, computational biology, clinical immunology, and data visualization will 1) improve the experience of end-users using nanowells to study cells from biopsies by increasing the number of samples each user can process through engineering and automation, by streamlining the process for extracting, integrating, analyzing and viewing data, and by enhancing the ability to recover rare cells; 2) validate modular nanowell-based operations for determining the types of cells present (cytometry) and their secreted proteins (microengraving) and the efficiencies of recovering cells and genes expressed relative to current standards; and 3) deploy the platform as a core facility at the Ragon Institute, making the technology broadly available for the first time to the community of end-users (scientists and physicians studying phenotypic diversity in clinical samples). The success of the project will yield a quantitative increase in the number of samples analyzed in nanowells per user, define protocols for executing assays comparable to conventional technologies, and establish a publicly-accessible platform for end-users, opening up new biology in all areas of human cellular disease and treatments.